Our long-term goal is to understand the molecular and cellular mechanisms for the generation of functional memory cytotoxic T lymphocytes (CTLs) for the purpose of developing more effective vaccines. Induction of protective memory CTLs is one of the major challenges in the development of effective vaccination against intracellular pathogens, such as HIV. However, the proteins required to generate functional memory CTLs are largely unknown. Biological function is based on proteins. We propose to identify the proteins involved in the regulation of memory CTL differentiation using high-throughput proteomics. We postulate that a limited number of proteins are responsible for memory differentiation, which can be identified by quantitative comparative proteomics. Our central hypothesis is that Key transcription factors related to survival are responsible for the regulation of memory CTL differentiation. We will utilize proteomic strategies to identify nuclear proteins that are potentially involved in the regulation of memory differentiation. In this R21 exploratory study, we will ask one major question: what are the most significant nuclear proteins whose abundances are altered when memory differentiation is regulated? First, we will seek to define the nuclear proteins differentially expressed during memory CTL differentiation during virus infection in animals. Second, through up regulation of memory CTLs through inhibition of mTOR by rapamycin, we will identify critical nuclear proteins whose abundances are further altered. To test our hypothesis, we will: 1) identify nuclear proteins potentially involved in memory CTL differentiation; 2) identify nuclear proteins potentially involved in the enhancement of memory CTL by rapamycin; 3) examine the transcription of potential targets identified in the first two aims. We expect that this work will lay the foundation for a future R01 application to study mechanisms for the generation of functional memory cytotoxic T lymphocytes, building on the leads revealed in this R21 work.